Compositions and methods for their preparation from lepidium

ABSTRACT

The invention relates to compositions that can be isolated from Lepidium plant material and to methods for their isolation. The compositions are useful for treating and preventing cancer and sexual dysfunction.

This application is a continuation of U.S. patent application Ser. No.09/878,141, filed Jun. 8, 2001, now abandoned, which is a divisionalapplication of U.S. patent application Ser. No. 09/261,806, filed Mar.3, 1999, now U.S. Pat. No. 6,267,995, the disclosure of which is hereinincorporated by reference in its entirety.

The present invention relates to compositions containing particularcomponents that can be obtained from a plant which can havepharmaceutical applications. More particularly, the plant genus isLepidium.

Lepidium meyenii, commonly called maca or Peruvian ginseng, is aperennial plant having a fleshy, edible, tuberous root. Another speciesis Lepidium peruvianum. The maca root is consumed for food and is alsoconsumed for its pharmacological properties; for example to enhancefertility. (See Leon, J., Economic Botany, 18:122-127(1964)) Maca hasalso been used to treat chronic fatigue. (Steinberg, P., PhilSteinberg's Cat's Claw News, Vol. 1, Issue 2, July/August (1995).

Johns, Ethnobiology, 1:208-212(1981), studied the biologically activearomatic glycosinolates glucosinolates present in the plant and alsoreported that benzyl isothiocyanate was the principal isothiocyanate inthe plant, with p-methoxybenzyl isothiocyanate being present inrelatively smaller amount. The role of these species in reproduction wasdiscussed.

Dini et al., Food Chem., 49:347-349(1994) described the total content ofcarbohydrates and amino acids (free and from protein hydrolysis) andlisted 20 saturated and unsaturated paraffinic acids (principallylinoleic, palmitic, and oleic acids) present in the plant. A sterolfraction containing five sterols (identified as their acetates) was alsoreported. An alkaloid fraction was reported but not characterized.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to an isolated,Lepidium-derived composition that is essentially free of cellulose andlignin and that has about 40% of polysaccharides that can be isolatedfrom Lepidium plant material (a Lepidium polysaccharide component). Thecomposition can also contain an aqueous component and a component,designated a Lepidium amino acid component, having amino acids that canbe isolated from Lepidium plant material and. Typically, the Lepidiumamino acid component has about 70% or more proline, 5% or more glutamicacid, and 5% or more valine. In certain embodiments, the composition hasabout 0.3% benzyl isothiocyante and about 0.5% of a component, called amacamide component, having amides of fatty acids that can be isolatedfrom Lepidium material. In an particular embodiment, the composition hasabout 45% or more of a Lepidium polysaccharide component. In anotherembodiment, the composition is combined with one or morepharmaceutically acceptable excipients.

In another aspect, the present invention relates to a composition havingabout 0.3% or more benzyl isothiocyanate, about 0.3% or more of amacamide component, about 1% or more of fatty acids that can be isolatedfrom Lepidium plant material (a Lepidium fatty acid component), andabout 0.15% of sterols that can be isolated from Lepidium plant material(a Lepidium sterol component). In a preferred embodiment, thecomposition has between about 2% and about 5% benzyl isothiocyanate,about 0.2% and about 1% Lepldium Lepidium sterol component, betweenabout 10% and about 20% Lepidium fatty acid component, and about 3% toabout 7% macamide component. In a particularly preferred embodiment, thecomposition has between about 5% and about 9% benzyl isothiocyanate,between about 1% and about 3% of Lepidium sterol component, betweenabout 20% and about 30% of a Lepidium fatty acid component, and about10% or more of macamide component. One or more of the foregoingcompositions can be combined with a pharmaceutically acceptableexcipient.

In another aspect, the invention relates to novel fatty acid amides:N-benzyl octanamide having the chemical formula C₁₅H₂₃NO,N-benzyl-16(R,S)-hydroxy-9-oxo-10E,12E,14E-octadecatrieneamide havingchemical formula C₂₅H₃₅NO₃, andN-benzyl-16(S)-hydroxy-9-oxo-10E,12E,14E-octadecatrieneamide having thechemical formula C₂₅H₃₅NO₃.

In yet another embodiment, the present invention relates to a process ofobtaining an isolated Lepidium-derived composition including the stepsof:

-   -   a) contacting Lepidium plant material with an aqueous solvent,    -   b) separating the contacted aqueous solvent from the Lepidiuin        plant material, and    -   c) concentrating the composition of step b) to isolate the        composition.        Preferably, the aqueous solvent is water. More preferably, the        aqueous solvent is a mixture of alcohol, preferably SDA, and        still more preferably, a mixture of 75 vol-% SDA and 25 vol-%        water.

In yet other embodiments, the process includes the further steps of:

-   -   d) applying the first Lepidium composition from step c) to a        reverse phase liquid chromatography column,    -   f) eluting the reverse phase chromatography column with a first        elution volume of aqueous solvent effective to elute a first        effluent from which a composition having about 45% or more        Lepidium polysaccharide component is isolated by, for example,        concentration.

In yet another embodiment, the invention process includes the furtherstep of g), eluting the reverse phase chromatography column with asecond elution volume of aqueous solvent to form a second effluent fromwhich the composition can be isolated by, for example, the step ofconcentrating the second effluent.

In a preferred embodiment, the second elution volume has two or moregradient volumes that are sequentially eluted and the resulting secondeffluent is fractionate collected as gradient fractions so that at leastone gradient fraction is collected for each gradient volume eluted. Thegradient fractions can be combined and concentrated to obtain anisolated Lepidium derived composition that has about 0.3% or more ofbenzyl isothiocyanate, about 0.15% or more Lepidium sterol component,about 1% or more Lepidium fatty acid component, and about 0.3% or moreof macamide component. Preferrably, each of the two or more gradientvolumes comprises a mixture of an alcohol and water and wherein thevol-% alcohol in the first gradient volume is about 20 vol-% or more andthe vol-% alcohol in any subsequent gradient volume is equal to orgreater than the vol-% alcohol in the immediately previously elutedgradient volume.

In yet another embodiment, the invention provides a process forproducing the composition having between about 5% and about 9% of benzylisothiocyanate, between about 1% and about 3% of Lepidium sterolcomponent, between about 20% and about 30% of Lepidium fatty acidcomponent, and d) about 10% or more of macamide component comprising thesteps of:

-   -   a) contacting Lepidium plant material with an aqueous solvent        comprising about 90 vol-% or more water,    -   c) concentrating the contacted aqueous solvent to make a residue        of Lepidium plant material,    -   d) contacting the residue of Lepidium plant material with        aqueous solvent comprising a mixture of an alcohol and water        having about 90 vol-% alcohol or more to form a liquor, and    -   e) concentrating the liquor to obtain the composition.

The preferred plant material in any embodiment is Lepidium meyenii.

In still another embodiment, the present invention relates to a methodof treating or preventing cancer in an animal, preferably a human, byadministering a cancer treating or preventing effective amount of any ofthe hereinabove described isolated Lepidium-derived compositions. Inanother embodiment, the present invention relates to a method fortreating sexual dysfunction in an animal, preferably a human, byadministering a sexual dysfunction treating or preventing amount of anyof the hereinabove described isolated Lepidium-derived compositions tothe animal. In a preferred embodiment, the animal is a female animal andthe sexual dysfunction is infertility. In a more preferred embodiment,the animal is a male animal and the sexual dysfunction is a sub-normallibido. In a yet more preferred embodiment, the animal is a male animaland the sexual dysfunction is impotence.

DETAILED DESCRIPTION

As used herein, the following terms have the following meanings.

Alcohol: The term alcohol refers to a lower aliphatic alcohol havingfrom one to six carbon atoms.

Aqueous component: This term refers to that part or portion of acomposition that is made-up of one or more aqueous solvents.

Aqueous solvent: The term aqueous solvent means water or a single phasehaving an organic solvent that is miscible with water. Examples ofmiscible organic solvents include but are not limited to methanol,ethanol, isopropanol, n-propanol, acetone, and acetonitrile. Othermiscible organic solvents are known to the skilled artesian.

Benzyl isothiocyante: includes benzyl isothiocynate and its methoxyderivatives.

Column volume: Column volume refers to the volume of the space definedby the inner surface of the chromatography column or chamber thatsurrounds the stationary phase or a reverse phase chromatography column.Column volume is abbreviated herein as CV.

Fractionate collecting: When used in connection with an effluent or agradient effluent, or a gradient fraction, the term fractionatecollecting denotes that the effluent or gradient effluent is segregatedinto at least two portions or aliquots.

Lepidium amino acid component: This term refers to that part or portionof a composition that is made-up of amino acids that can be isolatedfrom Lepidium plant material.

Lepidium composition: A Lepidium composition is a composition having atleast one of a Lepidium amino acid component, a Lepidium fatty acidcomponent, a Lepidium polysaccharide component, or a macamide component.

Lepidium fatty acid component: This term refers to that part or portionof a composition that is made-up of fatty acids that can be isolatedfrom Lepidium plant material.

Lepidium plant material: As used herein, Lepidium plant material refersto plant matter from any part of a plant of the genus Lepidium. Examplesof Lepidium plant matter include, but are not limited to, matter fromLepidium meyenii and Lepidium peruvianum.

Lepidium polysaccharide component: This term denotes that part orportion of a composition that is made-up of polysaccharides that can beisolated from Lepidium plant material.

Lepidium sterol component: As used herein, the term refers to that partor portion of a composition that is made-up of sterols that can beisolated from Lepidium plant material.

Macamide: Means amides and N-substituted amides of fatty acids that canbe isolated from Lepidium plant material.

Percent (%): Unless otherwise limited or modified, percents andpercentages described herein are on a weight basis. The chemicalcomposition of plant material from a particular plant species varieswith, for example, the conditions under which the plant is grown (forexample soil and climate). A particular compound or mixture of compoundscan exhibit pharmacological efficacy over a readily ascertainable rangeof composition and dosage. Therefore, it is understood that thepercentages recited throughout are meant to include such variationsoutside the stated percentage or percentage ranges as would beanticipated by the skilled artesian.

Substantially Free of cellulose: Means having 5% or less of celluloseand lignin combined.

SDA: Means special denatured alcohol, typically a mixture of 97% to 95%ethanol with 3% to 5% of methanol or coal tar.

First Lepidium compositions of the invention can be obtained from anextraction composition in a first process. Starting material for a firstprocess is Lepidium plant material, preferably Lepidium meyenii.Lepidium plant material, for example the root of Lepidium meyenii, isreduced in size to pieces having nominal dimensions between about 0.1 mmand 30 mm. The pieces of Lepidium plant material are contacted withaqueous solvent. The contacting in this or any embodiment may be by anysuitable means as are known in the art; for example, percolation, vatextraction, counter current extraction, and the like. The contacting isfor a time from about 2 hr. to about 18 hr. The contacting is carriedout at a temperature above the solidification temperature (or whereapplicable the phase separation temperature) but below the boiling pointof the aqueous solvent. Typically, the contacting is conducted from 20°C. to 75° C., with 40° C. to 50° C. being preferred. After thecontacting, the aqueous solvent, which is an extraction compositioncontaining a first Lepidium composition, is separated from residualplant material and the extraction composition is concentrated until theextrtaction composition has a solids component generally of at leastabout 70%, of which typically 40% is a Lepidium polysaccharidecomponent. In this or any embodiment, the concentration can be by any ofthe means as are known in the art such as evaporation, distillation, andlyophilization, to mention a few.

First Lepidium compositions generally have 30% or more, preferably about40% or more, of a Lepidium polysaccharide component and about 1% or moreof a macamide component. Typically, 70% or more of the polysaccharideunits of a polysaccharide of a Lepidium polysaccharide component aresucrose units. A macamide component includes N-benzyl octanamide(macamide A), N-benzyl-16-hydroxy-9-oxo-10E,12E,14E-octatrieneamidesN-benzyl-16-hydroxy-9-oxo-10E,12E,14E-octadecatrieneamide (macamide B),and N-benzyl-9,16 -dioxo-10E,12E,14E-octadecatrieneamide (macamide C).First Lepidium compositions further contain about 8% or more of aLepidium amino acid component. The Lepidium amino acid component of afirst Lepidium composition typicallylas typically has 70% or moreproline, 5% or more glutamic acid, and 5% or more valine. First Lepidiumcompositions also generally contain up to 1% of benzyl isothiocyanate.First Lepidium compositions can also contain about 0.5% to about 1.5%Lepidium fatty acid component. First Lepidium compositions of thepresent invention are useful for their nutritional value and are usefulfor the treatment or prophylaxis of carcinomas. First Lepidiumcompositions are also useful for treating sexual dysfunction inparticular sub-normal libido and impotence in males and infertility inwomen.

In a first process of the present invention, the composition of theextraction composition and the composition of the first Lepidiumcomposition obtained therefrom can be changed by changing the aqueoussolvent. When the aqueous solvent is an alcohol-water mixture having 75vol-% SDA, the first Lepidium composition generally contains about 0.8%or more benzyl isothiocyanate and a Lepidium fatty acid component ofabout 1% or more. When the aqueous solvent is 90 vol-% or more water,the first Lepidium composition has less than about 0.1% of benzylisothiocyanate and less than about 2% Lepidium fatty acid component.

In a preferred embodiment, a first Lepidium composition having aLepidium polysaccharide component of about 45% or more, a Lepidium fattyacid component between about 1% and about 2%, and less than about 1%each Lepidium sterol component and benzyl isothiocyanate is obtained bya suitably adapted first process that includes a reverse phase liquidchromatography process adapted to elute a first effluent containing afirst Lepidium composition. In reverse phase liquid chromatography(RPLC), the column packing (stationary phase, or adsorbent) isnon-polar, typically having a dipole moment of about 3 or less. Silicagel that has been treated to provide it with a bonded surface layer thatis paraffinic in nature is an example of a useful stationary phase forreverse phase chromatography. Silica gels having permanently bonded C₈to C₁₈ alkyl groups are commercially available as a stationary phase.For example, WP-Octadecyl from J. T. Baker Corp, Phillipsburg, N.J.,08865. Reverse phase liquid chromatography columns are eluted witheluents of decreasing polarity which causes the more polar compoundsloaded on a column to elute first.

Reverse phase liquid chromatography stationary phases of organicmaterial are also known. Polymers of vinyl aromatic compounds, forexample styrene, that are crosslinked with polyvinylic aromatichydrocarbons, for example divinyl benzene, can be used as stationaryphases for reverse phase liquid chromatography. These organic polymericstationary phases are made by processes that yield small, extremelyrigid, macroreticular particles. Crosslinked acrylic polymers are alsouseful as stationary phases for reverse phase liquid chromatography, asare polyvinyl alcohols (alkylated or non-alkylated). Suitable stationaryorganic phases for RPLC are commercially available. For example,styrenic and acrylic stationary phases are available from the Rohm andHaas Company, Philadelphia, Pa., under the trade name Amberlite®.Styrenic stationary phases are also available under the trade nameAmberchrom® from Tossohass, Montgomeryville, Pa. Polyamide resins (e.g.nylons), polyester resins, and phenolic resins are also usefulstationary phases for the reverse phase chromatography processes of thepresent invention.

Many organic solvents are suitable mobile phases, or eluents, forreverse phase liquid chromatography. Lower alcohols, such as methanol,ethanol and propanol as well as nitrites such as acetonitrile, aresuitable as organic eluents. Lower aliphatic ketones such as acetone,methyl ethyl ketone, and diethyl ketone, as well as cyclic ethers suchas tetrahydrofuran, can also be used. Dimethyl formamide, dimethylsulfoxide, and alkyl esters of acetic acid such as ethyl acetate canalso be used. Mixtures of such solvents in various proportions can beused when it is desired to elute or wash the column with solvents ofvarying polarity. Applicants have found that aqueous solvents that are mof water and an alcohol, for example, methanol, ethanol n-propanoliso-propanol n-butanol, and n-and sec-hexanol are particularly useful asmobile phases or eluents for the RPLC processes of the presentinvention, which in certain embodiments are carried out using an eluentof variable composition. Thus, an elution volume which is a volume ofaqueous solvent applied to the column, can be a gradient eluent havingtwo or more gradient volumes, the composition of which can be the sameor different, or the compositon of the gradient eluent can be variedcontinuously during elution. The composition of the elution volume thatis a gradient eluent can vary step-wise, linearly, sigmoidally,exponentially, logarithmically, parabolically, or hypyperbolicallyduring elution. The limits of concentration of gradient eluents aredetermined by the concentration of polar organic solvent necessary toelute products from the stationary phase and by the requirement that thepolar organic solvent be miscible to form a single phase at the requiredconcentration.

In certain embodiments of the present invention the initial alcoholconcentration in the elution volume is 10 volume percent (10 vol-%) orless and is increased as separation and purification proceeds.

The reverse phase liquid chromatography systems used to practice thepresent invention may be either preparative or analytical. Preparativecolumns have larger loading capacity and are typically larger in size.

With regards to the dimensions of the reverse phase liquidchromatographic column, the loading of the column, the temperature, andflow rate, one skilled in the art will know to vary these parametersbased primarily upon practical considerations known in the art. Forexample, flow rates of the eluent are adjusted according to the columndimensions, the degree of separation desired, the particle size of thestationary phase, and the back pressure in the column. The separation istypically carried out at 20° C. to 30° C. However, a temperature up toabout 45° C. can be used. The separation may be carried out at highpressure (500-200 psi) or moderate pressures (100-500 psi) or,preferably, at lower pressures (10-100 psi).

Prior to use, the reverse phase liquid chromatography column can beconditioned by eluting the column with a conditioning volume of aconditioning liquid, preferably an aqueous solvent, more preferablywater. The conditioning volume is preferably between about 1 and about10 column volumes.

The material to be treated is applied to the preferably conditionedreverse phase chromatography column as a solution, a slurry, or aloading concentrate obtained by evaporating an aqueous solvent,preferably alcohol, from an extraction composition containing theproduct. If the product to be treated is solid, it may be mixed with asuitable solid carrier, for example treated or untreated silica gel, andthe solid mixture placed on top of the solid support. Loading of thecolumn is accomplished by eluting the solution, slurry, or loadingconcentrate through the column; or, when the product to be treated isadmixed with silica gel, by eluting the column with a loading elutionvolume. Preferably, elution of the solution, slurry, loadingconcentrate, or loading elution volume is followed by elution with awashing elution volume comprising an aqueous solvent having the samecomposition as the aqueous solvent of the solution, slurry, or loadingconcentrate used to load the column stationary phase. The washingelution volume, when one is used, is preferably between about 1 andabout 10 column volumes.

Starting material for this adapted first process is a first Lepidiumcomposition made by the previously described first process in which theaqueous solvent is an alcohol—water mixture having between about 65vol-% and about 85 vol-%, preferably about 75 vol-% of alcohol,preferably SDA. The stationary phase of the RPLC column is a styrenicresin, preferably a crosslinked styrene—divinylbenzene resin such asAmberlite® XAD-16HP available from Rohm and Haas. The first Lepidiumcomposition from a first process, combined with water (5-7 L per kg offirst composition), is eluted through an RPLC column to apply the firstcomposition to the column. The column is then eluted with an elutionvolume effective to elute a first effluent containing a first Lepidiumcomposition, the solids component of which includes about 45% or more ofa Lepidium polysaccharide component. Typically the elution volumecontains aqueous solvent that is preferably water or an alcohol—watermixture having at least about 90 vol-% water and the elution volumeamounts generally to 4 to 7, preferably 6, column volumes. The effluentis collected and concentrated to yield a first Lepidium compositionhaving a Lepidium polysaccharide component of about 45% or more.

The first process can be further adapted to produce a second effluent byincluding, after elution of a first elution volume, the step of elutinga second elution volume that includes an aqueous solvent. SecondLepidium composition is obtained by concentrating the second effluent.In this or any other embodiment, the concentrating can be by anysuitable means as know in the art such as evaporation, distillation,lyophilization, and the like. Generally, the second elution volume istypically to 4 to 10 column volumes. The second effluent contains asecond Lepidium composition having between about 0.3% and about 12%,preferably between about 2% and about 10%, more preferably between about5% and about 10% of benzyl isothiocyanate; between about 0.3% and about2.7%, preferably between about 1% and about 2.5% of a Lepidium sterolfraction; and between about 10% to about 65%, preferably between about10% and about 25% of a Lepidium fatty acid component.

In one embodiment of the adapted first process, the second elutionvolume has at least two gradient volumes, each of which contains anaqueous solvent. The identity or composition of the aqueous solvent ineach of the two or more gradient volumes can be the same or it can bedifferent. Preferably it is different. The second effluent resultingfrom the elution of the second elution volume can be fractionatecollected into at least as many gradient fractions as there are gradientvolumes eluted. The fractionate collected gradient fractions can becombined, or they can be maintained separately. The gradient fractionscan be concentrated to obtain second Lepidium compositions. It will beapparent to one skilled in the art that the identity and amounts ofconstituents of second Lepidium compositions made by this process can bevaried by varying the number and composition of gradient volumes eluted,the number and volume of gradient fractions that are fractionatecollected, and the manner in which gradient fractions are combined.

In a preferred embodiment of the adapted first process, the secondelution volume is an alcohol—water mixture having 70 vol -% andpreferably 80 vol-% or more alcohol, preferably SDA. The second Lepidiumcomposition obtained in this process contains between about 0.2% andabout 10%, preferably between about 2% and about 5%, of benzylisothiocyanate; between about 0.15% and about 3%, preferably betweenabout 0.2% and about 1%, of a Lepidium sterol component; between about1% and about 65%, preferably between about 10% and about 25% of aLepidium fatty acid component; and between about 0.3% and about 0.5% ofa macamide component, wherein the macamide component comprises about 30%or more macamide B and 20% or more macamide C. Second Lepidiumcompositions typically have less than about 5%, preferably less thanabout 1%, of either a Lepidium polysaccharide component or a Lepidiumamino acid component.

A second Lepidium composition can also be obtained from a secondextraction composition from a second process. In a second process,residual Lepidium plant material that remains after separating the firstextraction composition is contacted in a second contacting step with anaqueous solvent, preferably a mixture of an alcohol and water havingabout 80 vol-% or more, preferably about 90 vol-% or more alcohol,preferably SDA, to produce a second extraction composition or liquor.The process of contacting in the second contacting step can be the sameas that in the first contacting step of a first process or it can bedifferent. The liquor is separated from residual Lepidium plant materialand concentrated to obtain a second Lepidium composition. The separatingcan be by any process known in the art; for example centrifugation,filtration, or decanting.

The second Lepidium composition obtained by a second process (MD-A) hasbetween about 5% and about 9% of benzyl isothiocyanate, a Lepidiumsterol component of between about 1% and about 3%, a Lepidium fatty acidcomponent between about 10% and about 20%, and a macamide component of10% or more. Generally, a Lepidium sterol component includes about 50%or more β-sitosterol and also contains stigmasterol and campesterol.Generally, a Lepidium fatty acid component contains between about 0.5%and about 1.2% 9,16-dioxo-10E,12E,14E-octadecatrieneoic acid betweenabout 1.5% and about 3.5% of16-hydroxy-9-oxo-10E,12E,14E-octadecatrieneoic acid between about 30%and about 35% linoleic acid, and between about 15% and about 22%linolenic acid. Generally, a Lepidium fatty acid component also containsoleic acid. In preferred embodiments, the second composition alsocontains about 0.2% or more of a macamide component, wherein themacamide component comprises 1% or less macamide A, about 10% or moremacamide B, and about 20% or more macamide C.

The present invention also provides a third Lepidium composition thatcontains a mixture of a pharmaceutically acceptable excipient with afirst Lepidium composition, a second Lepidium composition, or with botha first and second Lepidium composition. Pharmaceutically acceptableexcipients are any materials that do not interfere with thepharmacological activity of the third composition or degrade the bodilyfunctions of the animal to which it can be administered, but facilitatefabrication of dosage forms or actual administration of the composition;for example by improving palatability of oral dosage forms. Examples ofpharmaceutically acceptable excipient include but are not limited tomaltodextrin, calcium phosphate, and fused silica. Pharmaceuticallyacceptable excipients also include flavorants.

Third compositions of the present invention can be made, for example, bycombining about 25% to about 50% (dry weight) of a first or secondLepidium composition of the present invention and about 75% to about 25%(dry weight) of one or more pharmaceutically acceptable excipients,combining this mixture with water (5 liter per kg of solids) andhomogenizing the mixture. A Silverson Model 14 RT-A homogenizer(Silverson Corporation, East Longmeadow, Ma.) is an example of anapparatus suitable for carrying-out the homogenization. The homogenizedcomposition is then dried to obtain a third Lepidium composition. Thedrying may be carried-out by any means as are known in the art; forexample spray drying, oven drying, rotary vacuum drying, orlyophilization.

In yet other embodiments, the present invention provides novel amides offatty acids, namely; N-benzyl octanamide (also called macamide A orMA-3), racemic and enantiomerically pureN-benzyl-16-hydroxy-9-oxo-10e,12e,14e-octadecatrienamideN-benzyl-16-hydroxy-9-oxo-10E,12E,14E-octadecatrienamide (also calledmacamide B or MA-S-4), and N-benzyl9,16-dioxo-10e,12e,14e-octadecatrienamideN-benzyl-9,16-dioxo-10E,12E,14E-octadecatrienamide (also called macamideC or MA-9). All of these N-substituted amides are members of the classmacamides, as that term is used herein. The N-benzyl amides of thepresent invention, which are useful in the prevention or treatment ofcarcinomas, can be synthetically prepared, or obtained from Lepidiumplant material, preferably Lepidium meyenii, by chromatographicprocesses. The chromatographic process is particular useful forobtaining enantiomerically pureN-benzyl-16(S)-hydroxy-9-oxo-10e,12e,14e-octadecatrienamideN-benzyl-16(S)-hydroxy-9-oxo-10E,12E,14E-octadecatrienamide.

N-benzyl amides of the present invention can be synthetically preparedby various methods (See Barstaw, L. E. et al., J. Org. Chem., 36,1305,(1971)). For example, N-benzyl amides of the present invention canbe made by refluxing the corresponding carboxylic acid and benzyl aminewith triphenylphosphene and bromotrichloromethane.

The N-benzyl amides of the present invention can be isolated fromLepidium plant material by c chromatographic process. Starting materialfor isolation of the N-benzyl amides of the present invention bychromatographic processes is a dry powder loading composition formed bymixing with silica gel (60-100 mesh) the residue from rotary vacuumconcentration of an ethyl acetate extract of an aqueous suspension of afirst Lepidium composition from a first process in which the aqueoussolvent contains a mixture of about 75% SDA and about 25% water. The drypowder loading composition is applied to a chromatography column that isthe eluted with a series of gradient volumes. The resulting gradienteluents are fractionate collected and compared by thin layerchromatography (TLC). Fractionate collected gradient eluents havingsimilar TLC patterns are combined and combined fractions are furthertreated by column chromatography.

The methods of preventing or treating carcinomas, libido-related malesexual dysfunction, male impotence, and muscle fatigue compriseadministering or dosing an effective amount of a composition, which canbe a third Lepidium composition, that contains a first or secondLepidium composition, or both. The meaning of effective amount will berecognized by clinicians but includes an amount effective to either (1)reduce the symptoms of the disease or condition sought to be treated orprevented (i.e. cancer, sexual dysfunction), (2) induce apharmacological change relevant to treating or preventing the diseasesought to be treated or to prevented, or (3) prevent the occurrence ofthe disease or condition.

The Lepidium compositions used in the method of the present inventioncan be administered by any route. Compositions of the present inventionare administered alone, or are combined with apharmaceutically-acceptable carrier or excipient according to standardpharmaceutical procedures. Preferably, Lepidium compositions areadministered orally as a third Lepidium composition. For the oral modeof administration, the compositions of the present invention are used inthe form of tablets, capsules, chewing gum, and the like. In the case oftablets, various disintegrants such as starch, and lubricating agentssuch as magnesium stearate and talc can be used.

Compositions of the invention can include pharmaceutically acceptableacid addition salts, particularly those obtained with mineral acids, forexample hydrochloric or hydrobromic acid. However, organic acids, forexample tartaric acid, can also be used.

The amount dosed will depend upon the composition used and the diseaseor condition to be treated or prevented. Generally, the compositions aredosed at between 0.1 g and 10 g per kg of body weight per day.

The present invention is illustrated by the following non-limitingexamples.

EXAMPLE 1

In this and other examples, HPLC analysis of Maca product were performedon a Hewlett Packard Series 1100 HPLC using an phenomenex, Luna C-8column. GC/MS analysis of Maca product was performed on an HP-5973 MSDusing a Supelco SAC-5 capillary column.

Roots of Lepidium meyenii (4.6 Kg) were cut to a nominal dimension ofabout 1 cm and contacted with an aqueous solvent (75 vol-% SDA and 25vol-% water; 25 L per kg root) at 45° C. by percolation. Decoction wasseparated from the plant material and concentrated to yield a firstcomposition (2.1 Kg), denoted NE, having a solids content of 77% (i.e.1.6 Kg on a dry basis). The solids component included, based on thesolids present; 0.89% benzyl isothiocyanate, 0.079% of a Lepidium sterolcomponent, 1.46% of a Lepidium fatty acid component, 8.72% of a Lepidiumamino acid component, and 41.9% of a Lepidium polysaccharide component.

EXAMPLE 2

Roots of Lepidium meyenii (500 g) were reduced in size to a nominaldimension of 0.5 cm and contacted with 14 L water by percolation. Theaqueous phase was separated from residual plant material and evaporatedto dryness to obtain 20 g of a tacky product. The tacky product had aLepidium sterol component of <0.01%, a Lepidium fatty acid component of<0.1%, a Lepidium amino acid component of 9%, and A Lepidiumpolysaccharide component of 44%.

EXAMPLE 3

The residual plant material from example 2 was contacted with 15 L of100% SDA by percolation to form a liquor. The liquor was separated andconcentrated to yield 10 g of a second Lepidium composition having 7.8%benzyl isothiocyanate, a Lepidium sterol component of 1.8%, a Lepidiumfatty acid component of 22%, and a macamide component of 12%. NoLepidium amino acid component was found in the composition.

EXAMPLE 4

A first Lepidium composition (1.6 Kg) obtained according to the processof example 1 (i.e. NE), was slurried with water (8.8 L per kg of dry NE)for about 30 min. The slurry was applied to a RPLC column (15 cm×100 cmpacked with Amberlite® XAD-16 (Rohm and Haas Co.) that had beenpreconditioned with 20 column volumes of water. The column was elutedwith a first elution volume (6 column volumes) of 100 vol-% water. Theresulting first effluent was collected and evaporated to dryness toyield 1.5 g of a first Lepidium composition having 0.15% benzylisothiocyanate, 0.07% of a Lepidium sterol component, 1.8% of a Lepidiumfatty acid component, 10% of a Lepidium amino acid component, and 48% ofa Lepidium polysaccharide component. The composition had less than 1% ofa macamide component.

EXAMPLE 5

The RPLC of example 4 was eluted with a second elution volume (6 columnvolumes) that was made-up of 100% SDA. The resulting effluent wascollected and evaporated to dryness to yield 162 g of a second Lepidiumcomposition having 4.1% benzyl isothiocyanate, 0.4% of a Lepidium sterolcomponent, 12% of a Lepidium fatty acid component, 4.4% of a macamidecomponent, and no Lepidium amino acid component or Lepidiumpolysaccharide component.

EXAMPLE 6

A first Lepidium composition (1.6 Kg NE on a dry basis), obtainedaccording to the method of example 1, was slurried with water (6.3 L perkg of first Lepidium composition) for about 30 min. The slurry wasapplied to a RPLC column (15 cm×100 cm) packed with Amberlite® XAD-16resin (Rohm and Haas Co) that had been preconditioned with about 20column volumes of water. The column was eluted with a first elutionvolume of 4 column volumes of water. The column was then eluted with asecond elution volume that was made-up of five gradient volumes, eachhaving a volume equal to 4 column volumes and each made-up of a mixtureof SDA and water. The gradient fractions had, respectively, 20 vol-%, 40vol-%, 60 vol-%, 80 vol-%, and 100 vol-% SDA. The gradient fractionscorresponding to each of the gradient volumes were fractionate collectedand analyzed (HPLC and GC). The gradient fractions were free of both anLepidium amino acid component and a Lepidium polysaccharide component.The gradient fractions contained other components as set-out below inTable 1.

TABLE 1 Content of Various Lepidium Components in Gradient fractions ofa RPLC Process. Fr*. Fr. Fr. Fr. Fr. M-F1 M-F2 M-F3 M-F4 M-F5 Benzyl 0.70.4 2.5 2.5 5.8 isothiocyanate - % Lepidium sterol 0.36 0.18 0.67 0.872.2 component - % Lepidium 0.29 0.14 0.54 0.70 1.8 fatty acidcomponent - % Macamide 0 0 0.2 1.6 0 component - % *Fr. = fraction

The gradient fractions were combined and concentrated to yield 125 g ofa second Lepidium composition having 2.3% benzyl isothiocyanate, 0.8% ofa Lepidium sterol component, 17.2% of a Lepidium fatty acid component,and 0.4% of a macamide component.

EXAMPLE 7

The tests were performed according to the MTT assay (See, Mosmann, T.,J. Immun. Meth. 65, 55(1983).

Cells were planted in 96 well flat bottom plates with low evaporationlids. Three cell lines per plate were seeded in 0.2 ml medium per well.Each cell line was planted at the optimum concentration for itsparticular growth rate: HT-29 and A-549, 5000 c/ml; MCF-7, 15000 c/ml;A498, 10,000 c/ml; PC-3, 15000 c/ml; and PACA-2, 10,000 c/ml. Productswere tested at various dilutions (at least ten) to determine the ED₅₀.

The results are given in table 2

TABLE 2 6-Cell Line Clinical Test Results, Expressed as ED⁵⁰ values (inμg/ml) Sample Kidney Prostate Pancreatic Lung Breast Colon Sample CodeDescription A-498 PC-3 PACA-2 A-549 MCF-7 HT-29 MA-1 24.88 85.77 17.6628.66 >100 54.65 MA-2 9.08 4.05 3.06 17.97 29.33 22.97 MA-3 5.83 6.424.33 7.57 28.45 19.02 macamide A MA-7 51.79 64.22 31.62 63.37 93.4961.55 SY- 197A Maca NE 55.85 94.57 37.60 55.99 97.51 64.17 M-F1Purified >100 >100 >100 >100 >100 49.24 Product Fraction #1 (20% SDA)M-F2 Purified >100 >100 >100 >100 >100 40.57 Product Fraction #2 (40%SDA) M-F3 Purified >100 >100 >100 >100 >100 40.57 Product Fraction #3(60% SDA) M-F4 Purified 38.29 29.59 29.43 17.03 34.41 6.10 ProductFraction #4 (80% SDA) M-F5 Purified 29.54 20.66 24.94 25.37 32.57 3.09Product Fraction #5 (100% SDA) M-F2-5 Purified >100 30.96 37.56 33.2656.18 41.08 Product Fraction #2~5 Adriamycin 3.59 × 10⁻³ 2.81 × 10⁻²5.22 × 10⁻³ 3.16 × 10⁻³ 1.07 × 10⁻³ 2.20 × 10⁻²

EXAMPLE 8

Ten healthy rats were dosed by oral gavage with 5 g of third Lepidiumcomposition per kilogram of body weight. The animals were observed forsigns of gross toxicity for 14 days. The body weights of the animalswere checked on the 7^(th) and 14^(th) days. Body weight data iscollected in Table 3. Gross necropsy findings at terminal sacrifice wereunremarkable.

TABLE 3 Individual Bodyweight and Dosage. Bodyweight (g) Animal No. SexInitial Day 7 Day 14 Dose* 5025 M 246 309 349 1.6 5026 M 236 297 332 1.65027 M 251 340 383 1.7 5028 M 263 341 379 1.7 5029 M 242 308 335 1.65030 F 197 230 253 1.3 5031 F 200 231 261 1.3 5032 F 185 227 247 1.25033 F 193 230 250 1.3 5034 F 186 231 249 1.2 *Administered as a 60% w/wsuspension in distilled water. Specific Gravity = 1.259 g/ml.

EXAMPLE 9

Sufficient mice (22±1.5 g) were dosed for 21 days with 1 g of either oftwo third Lepidium compositions. One group (group I) was dosed with athird Lepidium composition made with a first Lepidium compositionobtained according to example 1. A second group (group II) was dosedwith a third Lepidium composition made with a second Lepidiumcomposition obtained according to example 5. One hour after the dosingon the 21^(st) day, each mouse was individually placed in water (25±2°C.) and observed. The elapsed time at which a remained submerged. Theresults are collected in Table 4.

TABLE 4 Muscle Fatigue Results. Animal Duration of Swim Time In NumberSeconds Group (n) (mean ± SD) p Control Group 15 110.07 ± 2.58 Group I15 124.07 ± 3.30 <0.01 Group II 15 144.13 ± 3.52 <0.01

EXAMPLE 10

Sufficient male mice (22±1.5 g; 15 per group) were dosed by oral gavagefor 21 days with 1 g/day of either of two third Lepidium compositions.Male mice in one group (group-I) were dosed with a third Lepidiumcomposition made with a first Lepidium composition obtained according toexample 1. Male mice in a second group (group II) were dosed with athird Lepidium composition made with a second Lepidium compositionobtained according to example 5. A control group received no thirdLepidium composition. Mice were ear-tagged or color coded foridentification. On the 21^(st) day, ½ hour after dosing, each male wasplaced in a cage with two mice and observed under darkroom conditions.The mice were observed for three hours and the number intromissions wererecorded. Intromission was indicated by a characteristic rearward lungeby the male terminating coitus. Results are summarized in table 5 below.

TABLE 5 Intromission Results. Number of Intromissions Group (mean ± SD)p Control 16.33 ± 1.78 Group I 46.67 ± 2.39 <0.01 Group II 67.01 ± 2.55<0.01

EXAMPLE 11

Testectomies were performed on a sufficient number of male rats usingpentobarbital, 45 mg/kg, as anesthetic. Rats were treatedpostoperatively for 3 days with sodium penicillin, 2,000 U/kg. Rats wereearcoded and colorcoded for identification and divided into 7 groups.Three groups of 10 rats each (Set A) were dosed by oral gavage for 21days with a third Lepidium composition made with a first Lepidiumcomposition obtained according to example 1. Rats in each of the threegroups received a different dosage. A second set of three groups of 10rats each (Set B) were dosed for 21 days with a third Lepidiumcomposition made with a second Lepidium composition obtained accordingto example 5. Rats in each of the three groups received a differentdosage. A control group of 10 rats received no third Lepidiumcomposition.

On the 21^(st) day, ½ hour after dosing, rats were restined and anelectric pulse of 20 V was applied to the penis using an electrode of aYSD-4G multifunction instrument. The time to achieve full erection wasmonitored. Results are collected in table 6 below.

TABLE 6 Incubation Period of Erection (IPE) in Testicle-Removed Ratswith Oral Administration of M-PE and MC-A PE. Incubation Period of DoseAnimal Number Erection in Seconds Group (mg/kg) (n) (mean ± SD) SurgicalGroup 10 137.4 ± 81.6  Set A Low dose 45 10 121.4 ± 51.3  Middle dose180 10 54.0 ± 25.8 High dose 1800 10 90.5 ± 80.2 Set B Low dose 45 1071.2 ± 32.  Middle dose 180 10 73.2 ± 39.  High dose 1800 10 80.9 ± 85.1

EXAMPLE 12

The N-benzyl amides of the present invention can be isolated fromLepidium plant material by chromatographic process. Staring material forisolation of the N-benzyl amides of the present invention bychromatographic processes is a dry powder loading composition formed bymixing with silica gel (60-100 mesh) the residue from rotary vacuumconcentration of an ethyl acetate extract of an aqueous suspension of afirst Lepidium composition from a first process in which the aqueoussolvent contains a mixture of about 75% SDA and about 25% water. The drypowder loading composition is applied to a silica gel column (130-270mesh) and the column is then eluted with, in sequence, five elutionvolumes, each about 8 column volumes that contain mixtures of n-hexaneand acetone in the following ratios (vol-% n-hexane:vol-% acetone):10:1, 5:1, 2:1, 1:1, and 0:1. The column effluent is divided into 18fractions. The 25^(th) through 27^(th) liter of effluent make-up a sixthfraction. The 41^(st) through 49½^(th) liter of effluent make-up a ninthfraction. The 63^(rd) through 70^(th) liter of effluent make-up atwelfth fraction.

N-benzyl octanamide can be obtained by chromatographic treatment of thesixth fraction by treating the sixth fraction on a silica gel columnusing a n-hexane—acetone mobile phase (3:1). N-benzyl octanamide can beisolated from the effluent by removing mobile phase from the effluent.

N-benzyl octanamide has the following physical and spectroscopicproperties: white powder, m.p.=76-78° C.; UVλ_(max)(MeOH)=213 nm(ε=11007). IR_(γKBr) IR υ_(KBr) (cm⁻¹): 3407, 2938, 2859, 1628, 1544,and 1451. Molecular Formula: C₁₅H₂₃NO. EI MS m/z [M⁺]: 162, 149, 148,106, 91, 77, and 57. ¹H and ¹³C NMR (CDCl₃): 2.17 (2H, t, J=7.2 Hz,H-2), 1,59 (sH, m, H-3), 1.28 (8H, m, H-4, 4, 6, 7′), and 5.65 (1H, brs, NH). The structure of N-benzyl octanamide can be represented, withoutregard to stereochemistry, as:

N-benzyl-9,16dioxo-10E,12E,14E-octadecatrieneamideN-benzyl-9,16-dioxo-10E,12E,14E-octadecatriene amide (MA-9,macamide C) can be isolated from the ninth fraction by fitherfurtherchromatographic treatnenttreatment of the fraction on a silica gelcolumn using 30 elution volumes of hexane/acetone as mobile phase.Fractions 15-20 (i.e. effluent from elution volumes 15-20) were furthertreated by preparative HPLC (Dynamax-60A column) usingacetonitrile—-water gradient eluent (5:95 to 95:5 vol. ratio,acetonitrile:water) to obtainN-benzyl-9,16-dioxo-10E,12E,14E-octadecatrienamide. The structure ofmacamide C can be represented, without regard to stereochemistry, as:

N-benzyl-9,16dioxo-10E,12E,14E-octadecatrienamideN-benzyl-9,16-dioxo-10E,12E,14E-octadecatrienamidehas the following physical and spectroscopic properties: Light yellowpowder, m.p.=115-116° C.; UVλ_(max)(MeOH)=317 nm (ε=13847). IR_(γKBr)(cm⁻¹) IR υ_(KBr) (cm⁻¹): 3297, 2930, 1715, 1680, 1637, 1602, 1544,1112, and 1003. Molecular formula (elemental analysis): C₂₅H₃₃NO₃. EI MSm/z, 395; [M+];: 366, 338, 260, 163, 106, 91, and 77. The structure ofmamamide B macamide B can be represented, without regard tostereochemistry, as:

N-benzyl-16(S)-hydroxy-9-oxo-10E,12E,14E-octadecatrienamide (MA-S-4,macamide B) can be isolated from the twelfth fraction by preparativeHPLC (Dynamax C-18 column) of the twelfth fraction using anacetonitrile—water gradient mobile phase (10:90 to 90:10 volume ratioacetonitrile:water).

N-benzyl-16(S)-hydroxy-9-oxo-10E,12E,14E-octadecatrienamide has thefollowing physical and spectroscopic properties: White powder, m.p.95-96° C., UVλ_(max) (MeOH)=314 nm (ε=36392). IR_(γKBr) IR υ_(KBr)(cm⁻¹): 3368, 2930, 2850, 1677, 1622, 1598, 1583, 1240, 1108, and 1057.Molecular formula (elemental analysis): C₂₅H₃₅NO₃. CI MS m/z, 397.

Experimental details are given below.

Contacting of Lepidium meyenii was carried out by percolation accordingto the following procedure. The roots were grounded into pieces shorterthan 3 cm in length. The ground material was then equally divided andloaded into three similarly-sized percolators. For the first cover, 75%SDA was loaded into each percolator at a ratio of 4.2:1 (volume ofsolvent in liter: weight of material in kg). After the solution had beencirculated for 5 hr. at room temperature, the extract was transferred toa still and concentrated in vacuo at a temperature below 65° C. For thesecond cover, fresh 75% SDA was added into percolator #1. After thesolution had been circulated for 5 hrs, the extract was transferred topercolator #2. After the solution in percolator #2 had been circulatedfor 5 hrs, the extract was transferred to percolator #3. The solution inpercolator #3 was circulated for 5 hrs and the extract was transferredto the same still (for the first cover) and concentrated in vacuo at atemperature below 65° C. The procedure as described above was repeatedthree more times to give a total of 5 covers, which were concentrateduntil the residue had a total solid dry base of 72.9%.

3.5 kg of the extract was suspended in 1.8 liter water, and extractedwith EtOAc (3×1000 ml). The combined extract was concentrated in vacuoat 40° C. to dryness (241 g). The residue was mixed with silica gel(60-100 mesh, 200 g) and air-dried, and then applied to a silica gelcolumn (7.5×60 cm, 800 g, 130-270 mesh) eluted with five 20 Lhexane/acetone gradient volumes (10:1-5:1-2:1-1:1-0:1, total volume 100l). Every 500 ml was fractionate collected and concentrated, a total of200 portions were collected. All portions were analyzed by thin layerchromatography (TLC) and portions having similar TLC patterns werecombined to give 18 fractions. The sixth fraction (1.8 g from 25^(th)through 27^(th) liters collected) was chromatographed over silica gel(60 g) using hexane/acetone (3:1, 2 l) as the mobile phase to give 25 mgof N-benzyloctanamide (macamide A or MA-3). The ninth fraction (3.4 g;41^(st) through 49^(th) liters collected) was initially separated oversilica gel column (120 g, 130-270 mesh) into 30 fractions (100 ml each).The fractions (frs 15-20) which mainly containedN-benzyl-9,16-dioxo-10E,12E,14E-octadecatrienamide (MA-9 or macamide C)were combined and subjected to preparative HPLC (column: Dynamax-60AC18, 2.14 I 25 cm i.d., 8 μm) using Acetonitrile/0.1% HOAc gradientsystem (0-60 min from 5% Acetonitrile to 95% Acetonitrile) as the mobilephase with a flow rate of 10 ml/min to give 10 mg of MA-9. The twelfthfraction (10 g) was chromatographed on silica gel eluting withCHCl₃/EtOAc (2:1, 200 ml per fraction, total 5 l). The fractions (10-15)mainly containedN-benzyl-16(S)-hydroxy-9-oxo-10E,12E,14E-octadecatrienamide (MA-S-4 ormacamide B) were combined and purified by preparative HPLC run on aDynamax C-18 column using an acetonitrile/0.1% HOAc gradient system(0-60 min from 10% Acetonitrile to 90% Acetonitrile) as eluent to give33 mg of MA-S-4.

MA-3 showed the similar UV and IR spectra as those ofN-benzyl-hexadecamide (MA-1) indicating that MA-3 was a fatty amide. The¹³C resonance of a carbonyl at δ173.2 supported the amide structure. The¹H signals at δ4.41 (2H, d, J=4.4 Hz), 5.71 (br s), 7.25 (5H), and ¹³Csignals at δ127.5 (1C, d), 127.8 (2C, d), 128.7 (2C, d), and 43.6 (1C,t) suggested that MA-3 had a same amine group as that of MA-1. Themolecular formula, C₁₅H₂₃NO, was derived from the EI mass spectrum andindicated five degrees of unsaturation. The benzene ring and the amidegroup accounted for all the five unsaturation degrees. Therefore, thefatty acid moiety was acyclic.

The ¹H NMR spectrum displayed a terminal methyl group at δ0.86, and theCOSY spectrum revealed coupling of this group with an unresolved eightproton proton complex at δ1.28. One methylene group was observed astriplet (J=7.2 Hz) at δ2.17, indicating that it was adjacent to thecarbonyl group. In the COSY spectrum, this methylene group was found tobe coupled to another methylene group at δ1.59 which in turn was coupledto the methylene signals at ca δ1.18. It was suggested that the fattyacid moiety had eight carbons. Treatment of this compound by 6N HClfollowed by CH₂N₂ gave methyl caprylate that was identified by GC-MSanalysis. Thus, the fatty acid moiety was unambiguously determined asoctanoyl. Therefore, MA-3 was identified as N-benzyl octanamide, alsodesignated herein as macamide A.

Compound N-benzyl-16(S)-hydroxy-9-oxo-10E,12E,14E, -octadecatrienamide(MA-S-4) showed maximum absorption at 314 nm (ε=36392), suggesting aconjugated trienone. It possessed a molecular formula C₂₅H₃₅NO₃ by massspectrum and the NMR data (Table 1), which requires eight unsaturationequivalents. The ¹H signals at δ4.73 (2H, d, J=6 Hz), 9.00 (br s, NH),7.35 (5H), and ¹³C signals at δ127.3 (1C, d), 128.1 (2C, d), 128.9 (2C,d), and 43.4 (1C, t) suggested that MA-S-4 had the same amine group asthat of MA-1 and MA-3. The ¹³C NMR spectrum revealed two carbonyl abenzene ring and three other olefins, accounting for all the degrees ofunsaturation and indicating MA-S-4 had an acyclic fatty acid moiety. TheDEPT and ¹³C NMR spectra showed the signals for total eighteen carbons,including one methyl, eight methylenes, seven methines, two quaternarycarbons for the fatty acid moiety. Thus, a C₁₈ fatty acid moiety wasproposed.

The COSY, with aid of TOSCY, assigned the all proton resonances. Theisolated terminal methyl group at δ1.07 (H-18) and methylene groups atδ2.45 (H-2) and 2.54 (H-8) were chosen as the starting points for theanalysis of COSY and TOCSY. The correlations between H-18 (δ1.07) andH-17 (δ1.74), H-17 and H-16 (δ4.41), H-16 and H-15 (δ6.25), H-15 andH-14 (δ6.71), H-14 and H-13 (6.82), H-13 and H-12 (δ6.44), H-12 and H-11(δ7.47), H-11 and H-10 (δ6.33) led to the assignment of the protonscontinuing from H-18 to H-10. The correlations between H-2 and H-3(δ1.63), H-3 and H-4 (δ1.24), H-8 and H-7 (δ1.83), H-7 and H-6 (δ1.34)led to the assignment of protons from H-2 to H-8 except for H-5. Theoverlaped proton systems at (δ1.24) contained four protons. Hence, twoprotons were assigned to H-5 (δ1.24). In HMBC spectrum, the long-rangecorrelations between H-2 (δ2.45) and C-1 (δ173.1), and H-8 (2.54) andC-9 (δ200.0) confirmed the assignment of H-2 and H-8. The ¹³C NMR datawere then assigned by HSQC spectrum. The ¹³C resonance of C-16 at δ72.7indicated that one hydroxy group was attached to C-16.

The large couplings between the olefinic protons (J_(10,11)=15.6 Hz,J_(12,13)=14.0 Hz, and J_(14,15)=15.6 Hz) revealed that three of thedouble bonds had E configurations. The absolute stereochemistry of C-16secondary alcohol was determined by optical rotations which were of thesame sign and magnitude ([α]_(D)+) as those of coalital ([α]_(D)+21°,C=0.63, Me₂CO) and 16(S)-hydroxy-9-oxo-10E,12E,14E,-octadecatrienoicacid ([α]_(D)+11.7°, C=0.2, Me₂CO), assigning a S configuration to C-16.The stereochemistry of coalital was determined by exciton chiralitymethod reported by Bernart et al., J. Nat. Prod. 56:245(1993)).

Therefore, MA-S-4 was determined to be N-benzyl-16(S)-hydroxy-9-oxo-10E,12E,14E-octadecatrienamide.

Compound N-benzyl-9,16-dioxo-10E,12E,14E-octadecatrienamide (MA-9) wasisolated as a light yellow powder, m.p. 115-6° C. The UV spectrum showedmaximum absorption at 317 nm (ε=13847), suggesting a conjugatedtrienone. The strong absorption bands at 2950 (aliphatic), 1715, 1680(conjugated ketone), 3297, 1640, 1545 (amide), and 1003 cm⁻¹ (transdouble bond) in the IR spectrum indicated that this compound was a fattyamide. The EI mass spectrum of MA-9 showed a molecular ion peak at m/z395 consistent with the molecular formula C₂₅H₃₃NO₃. The ¹³C resonanceof the carbonyl carbon at δ173.3 favored that MA-9 was a fatty amide.

The ¹H signals at δ4.62 (2H, d, J=4.4 Hz), 8.83 (br s), 7.30 (5H), and¹³C signals at δ127.6 (1C, d), 128.4 (2C, d), 129.2 (2C, d), and 43.6(1C, t) suggested that MA-9 had a same amine group as those of MA-1,MA-3 and MA-S-4. The intense fragment ion peaks at m/z 91 and 106supported the above inference.

All proton and carbon signals of the fatty acid moiety wereunambiguously assigned using COSY, TOCSY, HMQC and HMBC techniques. Theisolated terminal methyl group at δ1.01 (H-18), methylene groups atδ2.38 (H-2) and 2.50 (H-8), and olefinic protons for H-10 at δ6.30 andH-15 at δ6.34 were chosen as the starting points for the analysis ofCOSY and TOCSY. The methyl group (Me-18) were coupled with H-17 (δ2.61).The correlations between H-2 (δ2.38) and H-3 (δ1.62), H-8 (δ2.50) andH-7 (δ1.23) were observed. The correlations between H-3 and H-4 (δ1.19),H-7 and H-6 (δ1.19) were observed. The proton complex at δ1.19 had sixprotons. The H-5 signals were regarded to account the left two protons.The correlations between H-10 (δ6.30) and H-11 (δ7.30), H-15 (δ6.34) andH-14 (δ7.30), H-11 and H-12 (δ6.68), H-14 and H-13 (δ6.68) confirmed thetriene structure.

In HMBC, the correlations between H-2 (δ2.33) and C-1 (δ173.3), H-8(δ2.56) and one ketone (C-9, δ200.5) confirmed the assignment of H-2 andH-8. The correlation between H-17 (δ2.61) and another ketone (C-16,δ200.3) confirmed the position of another ketone at C-16. The positionsof the two ketone groups were supported by the daughter ion peaksobserved in the EI mass spectrum. The ions at m/z 366 and 338 derivedfrom the cleavage of C-17-C16 bond, and C-16-C-15 bond, respectively,indicated that a ketone was located at C-16. The ions at m/z 135, 163and 260 derived from the rupture of C-9-C-10 bond and C-8-C-9 bond,respectively, revealed another ketone at C-9.

The double bonds at C-10 and C-14 were determined to be E configurationsbased on the large coupling constants (J_(10,11)=15.3 Hz, andJ_(15,14)=15.3 Hz). Because the multiplets of H-11, H-12, H-13 and H-14are not first order, and may be AA′BB′ system, they do not displaysubstantive couplings each other. The configuration of the double bondat C-12 could not be determined by coupling constant. However, it mightbe suggested to be E configuration based on the inference that MA-9 be aoxidized product of MA-S-4.

Therefore, MA-9 was determined to beN-benzyl-9,16-dioxo-10E,12E,14E-octadecatrienamide. MA-9 is a newcompound and named macamide C.

1. A N-benzyl-16(R,S)-hydroxy-9-oxo-10E,12E,14E-octadecatrieneamidehaving a chemical formula C₂₅H₃₅NO₃.
 2. The An isolated compound ofclaim 1, wherein the compound isN-benzyl-16(S)-hydroxy-9-oxo-10E,12E,14E-octadecatrieneamide having achemical formula C₂₅H₃₅NO₃.
 3. A An isolated compoundN-benzyl-9,16-dioxo-10E,12E,14E-octadecatrieneamide having a chemicalformula C₂₅H₃₅NO₃.
 4. A composition comprising a mixture of about 0.3%or more of the isolated compound of claim 2 and a pharmaceuticallyacceptable excipient.
 5. The composition of claim 4 further comprising apharmaceutically acceptable salt.
 6. The isolated compound of claim 2,wherein said isolated compound is a dry powder.
 7. A compositioncomprising a mixture of about 0.3% or more of the isolated compound ofclaim 3 and a pharmaceutically acceptable excipient.
 8. The compositionof claim 7 further comprising a pharmaceutically acceptable salt.
 9. Theisolated compound of claim 3, wherein said isolated compound is a drypowder.